ABSTRACT Glioblastoma multiforme (GBM) is the most lethal primary brain cancer, with standard treatments based on surgery, radiotherapy, and chemotherapy promoting an overall survival of approximately 15 months. This has led to a resurgence of interest in immune-based approaches. While augmenting immunity has been successful in other malignancies like melanoma and leukemia/lymphoma, their efficacy in treatment of brain tumors has been very limited. Tumor antigen heterogeneity, limited number of infiltrating lymphocytes at the tumor site, and failure of checkpoint inhibitor drugs to cross the blood brain barrier all represent major obstacles to effective immune-therapies in GBM. Altered immunity in patients with GBM further contributes to the poor prognoses of these tumors and their relative resistance to vaccine and checkpoint inhibitors. Utilizing healthy donor immune cells in the form of adoptive cell therapy may offer a more effective alternative. However, limited information is available on the properties of effector populations that would exert effective anti-tumor activity in the brain. Pioneering work by Dr. Rick Jones at Johns Hopkins University utilizing haploidentical donor transplant (haplo-BMT) for brain tumors offers an opportunity to test kinetics of infused healthy donor immune cells in this patient population, providing a glimpse of what donor immune cells can do against brain tumors. Previous experiences with haplo-BMT suggest that effector cells of the innate immunity ? natural killer cells (NKs) and invariant natural killer T cells (iNKTs) ? mediate anti-tumor activity with decreased frequency of relapse. Innate immune cells are also potentially advantageous in the brain tumor setting, which affords very little tolerance for the adverse events associated with (for example) CD19-CAR T cell based-therapies. Finally, both iNKT and NK cells can actively migrate to the site of GBM following the CCL2 gradient, the chemokine released by tumor cells and the surrounding tumor associated macrophages (TAM). Dr. Savoldo at University of North Carolina has unique expertise in the development of chimeric antigen receptor (CAR) transduced T cells and iNKT cells and Dr. Bollard at Children's National has extensive experience with the genetically modified T cells and NK cells. Hence, in this collaborative proposal we hypothesize that nontolerized innate immune cells (NKs and iNKTs) derived from healthy donors will promote anti- tumor immunity in patients GBM, and may be developed as effective cellular therapies. This overarching hypothesis will be tested in 3 Specific Aims where Dr. Jones (Aim 1) will compare the cellular signatures of iNKTs and NK cells isolated from healthy donors versus patients with GBM. In Aim 2, Dr. Savoldo and the UNC team will evaluate the anti-tumor efficacy of healthy donor iNKT and NK cells modified to express an anti- B7H3 chimeric antigen receptor (CAR) and in Aim 3, Drs. Bollard and Cruz at CNMC overcome the inhibitory tumor microenvironment in GBM by targeting immune suppressive tumor associated macrophages (TAMs) and TGF?.